Controlled Cycling and Quiescence Enables Efficient HDR in Engraftment-Enriched Adult Hematopoietic Stem and Progenitor Cells

Jiyung J Shin,Markus S Schröder,Francisco Caiado,Stacia K Wyman,Nicolas L Bray,Matteo Bordi,Mark A Dewitt,Jonathan T Vu,Won-Tae Kim,Dirk Hockemeyer,Markus G Manz,Jacob E Corn

doi:10.1016/j.celrep.2020.108093

Abstract

SummaryGenome editing often takes the form of either error-prone sequence disruption by non-homologous end joining (NHEJ) or sequence replacement by homology-directed repair (HDR). Although NHEJ is generally effective, HDR is often difficult in primary cells. Here, we use a combination of immunophenotyping, next-generation sequencing, and single-cell RNA sequencing to investigate and reprogram genome editing outcomes in subpopulations of adult hematopoietic stem and progenitor cells. We find that although quiescent stem-enriched cells mostly use NHEJ, non-quiescent cells with the same immunophenotype use both NHEJ and HDR. Inducing quiescence before editing results in a loss of HDR in all cell subtypes. We develop a strategy of controlled cycling and quiescence that yields a 6-fold increase in the HDR/NHEJ ratio in quiescent stem cells ex vivo and in vivo. Our results highlight the tension between editing and cellular physiology and suggest strategies to manipulate quiescent cells for research and therapeutic genome editing.

Highlights

CRISPR-Cas genome editing has emerged as a powerful tool that enables fundamental research into genotype-phenotype relationships and holds great promise for the treatment of genetic disease (Doudna and Charpentier, 2014; Fellmann et al, 2017; Sternberg and Doudna, 2015)
We used a potent single guide RNA we previously found to efficiently edit human CD34+ HSPCs at the hemoglobin beta (HBB) locus and an single-stranded oligodeoxynucleotides donor template designed to modify the causative HBB mutation involved in sickle cell disease (SCD) (Figure S1A; Cradick et al, 2013; DeWitt et al, 2016)
After editing bulk CD34+ HSPCs, we measured the efficiency of homology-directed repair (HDR) and non-homologous end joining (NHEJ) in immunophenotypically sorted hematopoietic stem cells (HSCs) (CD34+ CD38À CD45RAÀ CD90+), multipotent progenitors (MPPs; CD34+ CD38À CD45RAÀ CD90À), and progenitors (CD34+ CD38+) (Figures 1A and 1B)

Summary

Introduction

CRISPR-Cas genome editing has emerged as a powerful tool that enables fundamental research into genotype-phenotype relationships and holds great promise for the treatment of genetic disease (Doudna and Charpentier, 2014; Fellmann et al, 2017; Sternberg and Doudna, 2015). In most human cell types, NHEJ is the primary repair mechanism throughout the cell cycle, whereas HDR occurs at a much lower rate and primarily happens in S/G2 phase due to template availability and to avoid inappropriate telomere fusion during mitosis (Branzei and Foiani, 2008; Essers et al, 2002; Hustedt and Durocher, 2016; Mao et al, 2008; Orthwein et al, 2014, 2015; Pietras et al, 2011; Saleh-Gohari and Helleday, 2004). The high levels of NHEJ and correspondingly low levels of HDR in primary cells have complicated both fundamental research and therapeutic applications that make use of genome editing. Primary hematopoietic stem cells (HSCs) ensure the lifelong production of all blood cells through their unique capacity to self-renew and to differentiate (Figure 1A). Inappropriate differentiation can lead to either the over- or under-production of blood components, causing disorders that range from immunodeficiency to cancer

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